New findings in the signaling pathways of cis and trans platinum Iodido complexes' interaction with DNA of cancer cells

We have selected a series of aliphatic amine platinum compounds bearing chloride and/or iodide as the leaving groups. The complexes' cytotoxicity and interaction with DNA indicated differences in the reactivity. Now, we are reporting on the analysis of their molecular mechanism of action on gastric cancer cells. Our data reveals differences between them. Chlorido drugs showed similar behavior to cisplatin; they both required p53 to induce apoptosis but only cis-ipa showed DNA damage requirement for apoptosis induction. On the contrary, cis and trans iodido induced cell death independent of p53 activity, and they induced cell death through Bid activation, so their toxicity could be enhanced in a combined treatment with novel Bcl-2 protein family inhibitors. We also report the structural features of the DNA adduct for one of the complexes by X-ray diffraction. These findings represent a step forward in the search for new platinum-derived agents more specific and effective in the treatment of cancerAuthors A.G.Q., M.C., and A.A.-V. received funding from MINECO CTQ2015-68779-R. I.S.P. received funding from P17-01401 (supported by FEDER funds) from Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, SpainNPL is a fellow of the Programa de Doctorado Biociencias Moleculares UAM, Madrid, Spain. NPL was supported by a fellowship Programa de Formación de Profesorado Universitario REF: FPU15/0466

XPA, XPC, and XPD modulate sensitivity in gastric cisplatin resistance cancer cells

Cisplatin is an election drug widely used in clinic for the treatment of advanced gastric cancer. However, the heterogeneity of the gastric tumors and its resistance to the drugs, make in some cases the response very low and the prognosis unpredictable. In this manuscript we aim to find the molecular processes involved in cisplatin-induced apoptosis in two gastric cancer cell lines with different sensitivity to the treatment: AGS and MKN45. The apoptosis induction is higher in MKN45 than in AGS cells in response to CDDP. The intrinsic apoptotic pathway study revealed that MKN45 cells undergo degradation of Mcl-1 together with an increase of Bid and Bad levels, which results in sensitivity to CDDP. In addition, DNA repair NER pathway is impair in MKN45 cells due to low levels of XPC and the absence of translocation of XPA and XPD to the nucleus after stimuli. Altogether, these results suggest that NER and Bcl-2 protein family proteins are potential targets to improve the response to cisplatin treatmentThis work was supported by PI1401495 and P17-01401 (supported by FEDER funds) from Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain and by CTQ2015- 68779R. JB-I was supported by a fellowship from Catedra Isaac Costero, funded by Banco Santander-UAM and Beca Nacional de Posgrado CONACYT. NP-L was supported by a fellowship Programa de Formación de Profesorado Universitario REF: FPU15/04669, Ministerio de Educación, Cultura y Deport

XPA, XPC, and XPD Modulate Sensitivity in Gastric Cisplatin Resistance Cancer Cells

Cisplatin is an election drug widely used in clinic for the treatment of advanced gastric cancer. However, the heterogeneity of the gastric tumors and its resistance to the drugs, make in some cases the response very low and the prognosis unpredictable. In this manuscript we aim to find the molecular processes involved in cisplatin-induced apoptosis in two gastric cancer cell lines with different sensitivity to the treatment: AGS and MKN45. The apoptosis induction is higher in MKN45 than in AGS cells in response to CDDP. The intrinsic apoptotic pathway study revealed that MKN45 cells undergo degradation of Mcl-1 together with an increase of Bid and Bad levels, which results in sensitivity to CDDP. In addition, DNA repair NER pathway is impair in MKN45 cells due to low levels of XPC and the absence of translocation of XPA and XPD to the nucleus after stimuli. Altogether, these results suggest that NER and Bcl-2 protein family proteins are potential targets to improve the response to cisplatin treatment

Regulatory effects of miR-19a on MAD2 expression and tumorigenesis in gastric cancer

Gastric cancer (GC) is worldwide the sixth most diagnosed and third leading cause of cancer deaths, with poor and late prognosis, probably due to post-surgery adjuvant treatment resistance and lack of a thorough panel of prognostic markers. We have previously shown that mitotic arrest deficient 2 (MAD2, encoded by MAD2L1), a key protein of the spindle assembly checkpoint, is relevant in GC cells; its interference impairs migration and growth, while its overexpression correlates with tumorigenesi

The angiotensin-(1-7)/Mas receptor a xis protects from endothelial cell senescence via klotho and Nrf2 activation

Endothelial cell senescence is a hallmark of va scular aging that pre disposes to vascular disease. We aimed to explore the capacity of the renin-angiotensin system (RAS) heptapeptide angiotensin (Ang)-(1-7) to counteract human endothelial cell senescence and to identify intracellular pathways mediati ng its potential protective action. In human umbilical vein endothelial cell (HUVEC) cult ures, Ang II promoted cell senescence, as revealed by the enhancement in se nescence-associated galactosidase (SA- -gal+) positive staining, total and telomeric DNA damage, adhesion molecules expression and human mononuclear adhesion to HUVEC monolaye rs. By activating the G-coupled receptor Mas, Ang-(1-7) inhibit ed the pro-senescence action of Ang II, but also of a non- RAS stressor such as the cytokine IL-1 . Moreover, Ang-(1-7) enhanced endothelial klotho levels, while klotho silencing resulted in th e loss of the anti-senescence action of the heptapeptide. Indeed, both Ang-(1-7) and recombinant klotho activated the cytoprotective Nrf2/heme-oxygenase-1 (HO)-1 pathway. The HO-1 inhibitor tin protoporphyrin IX prevented the anti-senescence action evoked by Ang-(1-7) or recombinant klotho. Overall, the present study ide ntifies Ang-(1-7) as an anti-senescence peptide displaying its protectiv e action beyond the RAS by consecutively activating klotho and Nrf2/HO-1. Ang-(1-7) mimetic dru gs may thus prove useful to prevent endothelial cell senescence and its re lated vascular complications

New Findings in the Signaling Pathways of cis and trans Platinum Iodido Complexes' Interaction with DNA of Cancer Cells

We have selected a series of aliphatic amine platinum compounds bearing chloride and/or iodide as the leaving groups. The complexes’ cytotoxicity and interaction with DNA indicated differences in the reactivity. Now, we are reporting on the analysis of their molecular mechanism of action on gastric cancer cells. Our data reveals differences between them. Chlorido drugs showed similar behavior to cisplatin; they both required p53 to induce apoptosis but only cis-ipa showed DNA damage requirement for apoptosis induction. On the contrary, cis and trans iodido induced cell death independent of p53 activity, and they induced cell death through Bid activation, so their toxicity could be enhanced in a combined treatment with novel Bcl-2 protein family inhibitors. We also report the structural features of the DNA adduct for one of the complexes by X-ray diffraction. These findings represent a step forward in the search for new platinum-derived agents more specific and effective in the treatment of cancer.Authors A.G.Q., M.C., and A.A.-V. received funding from MINECO CTQ2015-68779-R. I.S.P. received funding from P17-01401 (supported by FEDER funds) from Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, SpainNPL is a fellow of the Programa de Doctorado Biociencias Moleculares UAM, Madrid, Spain. NPL was supported by a fellowship Programa de Formación de Profesorado Universitario REF: FPU15/04669

Targeting MAD2 modulates stemness and tumorigenesis in human Gastric Cancer cell lines

[Rationale]: Gastric cancer (GC) is a solid tumor that contains subpopulations of cancer stem cells (CSCs), which are considered drivers of tumor initiation and metastasis; responsible for therapeutic resistance; and promoters of tumor relapse. The balance between symmetric and asymmetric division is crucial for stem cell maintenance. The objective of this study is to evaluate the role of MAD2, a key protein for proper mitotic checkpoint activity, in the tumorigenesis of GC.[Methods]: Gastric cancer stem cells (GCSCs) were obtained from MKN45, SNU638 and ST2957 cell lines. Pluripotency and stemness markers were evaluated by RT-qPCR and autofluorescence and membrane markers by flow cytometry. Relevant signal transduction pathways were studied by WB. We analysed cell cycle progression, migration and invasion after modulation of MAD2 activity or protein expression levels in these in vitro models. In vivo assays were performed in a nude mouse subcutaneous xenograft model.[Results]: We found that NANOG, CXCR4 and autofluorescence are common and consistent markers for the GCSCs analysed, with other markers showing more variability. The three main signalling pathways (Wnt/β-catenin; Hedgehog and Notch) were activated in GCSCs. Downregulation of MAD2 in MKN45CSCs decreased the expression of markers CXCR4, CD133, CD90, LGR5 and VIM, without affecting cell cycle profile or therapy resistance. Moreover, migration, invasion and tumor growth were clearly reduced, and accordingly, we found that metalloprotease expression decreased. These results were accompanied by a reduction in the levels of transcription factors related with epithelial-to-mesenchymal transition.[Conclusions]: We can conclude that MAD2 is important for GCSCs stemness and its downregulation in MKN45CSCs plays a central role in GC tumorigenesis, likely through CXCR4-SNAI2-MMP1. Thus, its potential use in the clinical setting should be studied as its functions appear to extend beyond mitosis.This work was supported by Fondo de Investigaciones Sanitarias (FIS), Instituto de Salud Carlos III, Spain, grants PI17-01401 (PR) and PI18/00757 (BS,Jr), (all co-financed through Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa”). NPL was supported by a grant FPU15/04669, funded by Ministerio de Educación, Cultura y Deporte, Spain. NPL is a fellow of the Doctoral Program in Molecular Biosciences, UAM, Madrid, Spain. BS,Jr was supported by a Ramón y Cajal Merit Award (RYC-2012-12104) from the Ministerio de Economía y Competitividad, Spain.Peer reviewe

Mad2 and BubR1 modulates tumourigenesis and paclitaxel response in MKN45 gastric cancer cells

Aneuploidy and chromosomal instability (CIN) are common features of gastric cancer (GC), but their contribution to carcinogenesis and antitumour therapy response is still poorly understood. Failures in the mitotic checkpoint induced by changes in expression levels of the spindle assembly checkpoint (SAC) proteins cause the missegregation of chromosomes in mitosis as well as aneuploidy. To evaluate the possible contribution of SAC to GC, we analyzed the expression levels of proteins of the mitotic checkpoint complex in a cohort of GC cell lines. We found that the central SAC proteins, Mad2 and BubR1, were the more prominently expressed members in disseminated GC cell lines. Silencing of Mad2 and BubR1 in MKN45 and ST2957 cells decreased their cell proliferation, migration and invasion abilities, indicating that Mad2 and BubR1 could contribute to cellular transformation and tumor progression in GC. We next evaluated whether silencing of SAC proteins could affect the response to microtubule poisons. We discovered that paclitaxel treatment increased cell survival in MKN45 cells interfered for Mad2 or BubR1 expression. However, apoptosis (assessed by caspase-3 activation, PARP proteolysis and levels of antiapoptotic Bcl 2-family members), the DNA damage response (assessed by H2Ax phosphorylation) and exit from mitosis (assessed by Cyclin B degradation and Cdk1 regulation) were activated equally between cells, independently of Mad2 or BubR1-protein levels. In contrast, we observed that the silencing of Mad2 or BubR1 in MKN45 cells showed the induction of a senescence-like phenotype accompanied by cell enlargement, increased senescence-associated β -galactosidase activity and increased IL-6 and IL-8 expression. In addition, the senescent phenotype is highly increased after treatment with PTX, indicating that senescence could prevent tumorigenesis in GC. In conclusion, the results presented here suggest that Mad2 and BubR1 could be used as prognostic markers of tumor progression and new pharmacological targets in the treatment for GC.This work was supported by the following grants: PS09/1988, 595, PI11 -00949 supported by FEDER funds and UAM-Santander CEAL-AL/2013-29. JBI is a fellow of the Programa de Doctorado Doble en Ciencias Biomédicas UNAM, Mexico City, Mexico/Biociencias Moleculares UAM, Madrid, Spain. JBI was supported by a fellowship from Cátedra Isaac Costero, funded by Banco Santander.Peer Reviewe